Fig. 6: A Lm lines secreting caspases-3 and -7-uncleavable LLO or caspase-3-uncleavable Iap are more virulent in HeLa cells.

Culture supernatants of ΔLLO Lm, transfected with indicated pIMK2-LLO constructs (A), were treated with caspase-3 or caspase-7 for 4 h (B). Secretion and caspase-mediated cleavage of LLO were assessed by immunoblots. Detection of Iap served as loading control in (A). C Human red blood cells were treated with serial dilutions (C) or caspase-treated (D) Lm supernatants containing LLOwt or LLOc3/7 for 15 min at 37 °C. Hemoglobin release was measured spectrophotometrically at 405 nm wavelength and average +/− SEM of three independent experiments is presented. E HeLa cells were infected with indicated Lm mutants +/− zVAD +/− TNF-α treatment before the intracellular bacterial load was assessed by growth curves assay. Arrows indicate the differences in delay times to the zVAD controls to reach the threshold OD of 0.1 that is plotted in (F). Average +/− SEM of the delay time normalized to zVAD controls in three independent experiments is shown. G Lm culture supernatants of lines secreting caspase-3-uncleavable (IAPc3) or wild-type Iap (IAPwt) were treated with caspase-3 for 4 h and cleavage of Iap was assessed by immunoblot. H HeLa cells were infected with indicated Lm mutants +/− TNF-α before the intracellular CFU were calculated from growth curves. Average +/− SEM of three independent experiments is shown. Significant differences between indicated groups are marked by asterisks. P-values are * < 0.05, **< 0.01 and ***< 0.005.